Techniques in the detection and characterization of phosphoramidate-containing proteins

Fujitaki, James M.; Smith, Rhona

doi:10.1016/0076-6879(84)07004-x

Cited by 80 publications

(61 citation statements)

References 32 publications

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“…In contrast, the GlcT phosphorylation signal disappeared completely after incubation with hydrochloric acid, whereas some phosphorylation of EIIBA Glc was still detectable under these conditions. The strong reduction of signal intensity of phosphorylated EIIBA Glc is in good agreement with the fact that one phosphate is present as a phosphoamidate (which is acid-labile), whereas the other is bound to a cysteine (this phosphate is stable under strongly acidic conditions) (31). Thus, we may assume that a phosphoamidate is present in GlcT.…”

Section: Phosphorylation Of Glct By Enzyme I and Hpr-thesupporting

confidence: 62%

Control of the Bacillus subtilis Antiterminator Protein GlcT by Phosphorylation

Schmalisch

Bachem

Stülke

2003

Journal of Biological Chemistry

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Bacillus subtilis transports glucose by the phosphotransferase system (PTS). The genes for this system are encoded in the ptsGHI operon, which is induced by glucose and depends on a termination/antitermination mechanism involving a riboswitch and the RNA-binding antitermination protein GlcT. In the absence of glucose, GlcT is inactive, and a terminator is formed in the leader region of the ptsG mRNA. If glucose is present, GlcT can bind to its RNA target and prevent transcription termination. The GlcT protein is composed of three domains, an N-terminal RNA binding domain and two PTS regulation domains, PTS regulation domain (PRD) I and PRD-II. In this work, we demonstrate that GlcT can be phosphorylated by two PTS proteins, HPr and the glucose-specific enzyme II (EII Glc ). HPr-dependent phosphorylation occurs on PRD-II and has a slight stimulatory effect on GlcT activity. In contrast, EII Glc phosphorylates the PRD-I of GlcT, and this phosphorylation inactivates GlcT. This latter phosphorylation event links the availability of glucose to the expression of the ptsGHI operon via the phosphorylation state of EII Glc and GlcT. This is the first in vitro demonstration of a direct phosphorylation of an antiterminator of the BglG family by the corresponding PTS permease.Glucose is the preferred source of carbon and energy for many bacteria. The sugar is transported into the cell and phosphorylated. The resulting glucose 6-phosphate can immediately feed into glycolysis. In Escherichia coli, Bacillus subtilis, and several other bacteria, glucose is taken up and concomitantly phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system (PTS).1 This system is made up of two general energy-coupling proteins, Enzyme I (EI) and HPr, and several multi-domain sugar specific permeases (Enzyme II, EII), which may exist as individual proteins or fused in a single polypeptide. In E. coli, the glucose-specific EII is composed of a membrane-bound protein comprising the actual transporter domain EIIC and the phosphotransfer domain EIIB. In addition, the EIIA domain is present as a cytoplasmic protein. In B. subtilis, all domains of the glucose permease are fused to form a single polypeptide with the domain arrangement EIICBA (1, 2).It was long considered that the genes encoding the components of the glucose PTS are constitutively expressed in bacteria. Although this is the case for the genes encoding the general proteins, ptsI and ptsH, the gene encoding EII Glc , ptsG, is induced by glucose in both E. coli and B. subtilis (1-3). In E. coli, regulation of ptsG expression is accomplished by the transcriptional repressor Mlc. In the presence of glucose, this repressor is sequestered by the non-phosphorylated EIICB Glc and, thus, is unable to repress ptsG transcription (3). In B. subtilis, glucose induction of ptsG expression is mediated by transcriptional antitermination. In the absence of glucose, transcription initiated at the ptsG promoter is terminated in the leader region of the mRNA. If glucose is present, an antiter...

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Section: Phosphorylation Of Glct By Enzyme I and Hpr-thesupporting

confidence: 62%

Control of the Bacillus subtilis Antiterminator Protein GlcT by Phosphorylation

Schmalisch

Bachem

Stülke

2003

Journal of Biological Chemistry

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“…The instability to both acid and base is a property of acyl phosphate bonds (8,18) (7,34). Also, these reduced amino 1, 3, and 5) or phosphorylated VirA681 with phosphorylated VirG (lanes 2, 4, and 6) were electrophoresed on a 12% SDS-polyacrylamide gel and transferred onto an Immobilon memnbrane.…”

Section: Resultsmentioning

confidence: 99%

Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG

et al. 1990

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Agrobacterium tumefaciens virulence genes are induced by plant signals through the VirA-VirG twocomponent regulatory system. The VirA protein is a membrane-spanning sensor molecule that possesses an autophosphorylating activity, and the VirG protein is a sequence-specific DNA-binding protein. In this report, we demonstrate that the VirG protein is phosphorylated by the VirA protein and that the phosphate is directly transferred from the phosphorylated VirA molecule (phosphohistidine) to the VirG protein. The chemical stability of the phospho-VirG bond suggested that the VirG protein was phosphorylated at the aspartate and/or glutamate residue. The phosphorylated VirG protein was reduced with tritiated sodium borohydride and subjected to proteolytic digestion with the Achromobater protease I enzyme. The resulting peptide fragments were separated by Cs reversed-phase high-pressure liquid chromatography, and the tritium-labeled peptide was sequenced. Amino acid sequence data showed that the aspartate residue at position 52 was the only site phosphorylated. Changing this aspartate into asparagine resulted in a nonphosphorylatable and biologically nonfunctional gene product. As a control, a randomly chosen aspartate was changed into an asparagine (position 72), and no effect on its phosphorylation or biological activity was observed. Unlike its homologs, including CheA-CheY, EnvZ-OmpR, and NtrB-NtrC, the phospho-VirG molecule was very stable in vitro. The possible implications of these observations and the function of VirG phosphorylation in vir gene activation are discussed.

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“…Also, we observed that the phosphate moiety on p38 tends to be stable in alkali at 55°C (1 M KOH for 1 h). However, at higher temperatures or with longer incubation time, the phosphate was labile (unpublished data), indicating that the products of p38 phosphorylation perhaps involved acyl phosphates (12). Further, we compared the sites of phosphorylation by peptide mapping (6).…”

Section: Resultsmentioning

confidence: 99%

Glucagon and p21 ras enhance the phosphorylation of the same 38-kilodalton membrane protein from rat liver cells.

Hegde

Das

1990

Mol. Cell. Biol.

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We had reported earlier the enhanced phosphorylation of a 38-kilodalton protein (p38) in rat liver plasma membrane by ras proteins. Now we show that glucagon increased the phosphorylation of the same protein. The nature and site(s) of phosphorylation were the same as those for the ras proteins. Both ATP and GTP could donate phosphate for the phosphorylation of p38. The stimulation of p38 phosphorylation by glucagon was guanine nucleotide dependent. This observation, together with our data on the stimulation of p38 phosphorylation by AIF4 , suggest the involvement of G proteins in the reaction. We also showed that glucagon stimulates the phosphorylation of p38 in vivo.The ubiquitous occurrence of the ras family of genes in eucaryotic organisms (2) and evidence for their expression in a number of human (9,36,38,41) and animal (1,2,5, 19,45) tumors as well as normal tissues (13,32,42) has resulted in considerable recent interest in the study of ras oncogenes. Although the ras proteins are known to bind GTP and GDP and have GTPase activity (16,31), the biological function of ras proteins remains unknown. GTPase-activating proteins have been shown to interact with ras proteins (31), but the exact role of ras in signal transduction still remains to be understood (24). Since phosphorylation is known to play a key role in signal transduction (7, 10, 22, 34), we had investigated the effect of ras proteins on the phosphorylation of plasma membrane proteins from rat liver and had reported increased phosphorylation of a 38-kilodalton protein (p38) present in the preparations (20).The ras proteins used in our experiments contained bound GDP (37). We had observed also (20) that GDP stimulates the phosphorylation of p38, although to a lesser extent compared with the stimulation observed in the presence of ras proteins. These observations suggested possible involvement of guanine nucleotides in the phosphorylation of p38. It was therefore of interest to check whether any of the factors which act on rat liver cells in a guanine nucleotide-dependent manner affected the phosphorylation of p38. We report here the effect of glucagon on the phosphorylation of p38 and show for the first time that the ras gene product and glucagon stimulate the phosphorylation of the target protein (p38) at an identical site(s).MATERIALS AND METHODS ras protein. The ras protein EC (normal human c-Ha-ras-1 with Gly-12, Ala-59, and Gln-61) expressed in Escherichia coli and purified to homogeneity (15) was provided by J. B. Gibbs of Merck Sharp & Dohme Research Laboratories.Plasma membrane preparations. Plasma membranes were isolated from livers of male Wistar rats by the procedure of Lesko et al. (27). This method yields highly purified plasma membrane preparations as judged by marker enzyme assays (40).Phosphorylation reactions. The in vitro phosphorylation reactions were performed as follows. To a 100-pl reaction mixture, 0.21 M D-mannitol, 0.07 M sucrose, 1 mM HEPES * Corresponding author.(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) at pH 7.5...

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Techniques in the detection and characterization of phosphoramidate-containing proteins

Cited by 80 publications

References 32 publications

Control of the Bacillus subtilis Antiterminator Protein GlcT by Phosphorylation

Control of the Bacillus subtilis Antiterminator Protein GlcT by Phosphorylation

Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG

Glucagon and p21 ras enhance the phosphorylation of the same 38-kilodalton membrane protein from rat liver cells.

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